This project will characterize the mitochondrial (mt) proteome of Trypanosoma brucei, and protein differences between life cycle stages. It will elucidate the functions of potentially all mt proteins and construct structural and functional protein interaction networks. This will extend understanding of mt physiology, and identify potential drug targets. The project will: 1. Identify as many proteins as possible in procyclic form (PF) mt and fractions thereof using high-throughput tandem mass spectroscopy (MS/MS), the SBEAMs data base/data management system, a suite of analytical software. and web interfaces. Fractionation will reduce sample complexity, enhance peptide detection, and identify sub-organellar locations and multi-protein complexes. 2. Determine associations among mitochondrial proteins by identifying the protein composition of purified ml complexes. The general functions of numerous proteins will be suggested from co-localization in complexes. 3. Construct the mitochondrial protein interactome. The datasets from aims 1 and 2 will be integrated with in silico analyses of extensive data from external sources using SBEAMS modules from which physical and functional interaction networks that will be generated, visualized using CYTOSCAPE, and disseminated via the web. This will generate a profile of T. brucei mt physiology and identify key steps in its pathways and processes. 4. Compare bloodstream and procyclic form mitochondrial proteomes. The presence of and composition of mt complexes will be compared between bloodstream (BF) and PFs. Differences in total mt proteins between these stages will be surveyed using procedures from aim 1. This will provide insights into BF physiology and be useful for predicting essential genes. 5.Examine the consequences of gene inactivation and drug treatment. The effect of inactivating genes with predicted critical functions will be analyzed. This will assess the validity of complex co-localizations, interaction maps, and assess the general functions of key proteins as well as drug susceptibility. Overall, this project will extend the TriTryp genome annotation, elucidate mt functional organization and its differences between life cycle stages, and aid the identification of mt drug targets and factors affecting drug susceptibility.